Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

Abstract

The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

“…Moreover, most transposon integrations corresponded to monomeric integrations and very low incidence of passive incorporation of vector backbone or SB transposase vector sequences (Table 1). Interestingly, all transgenic pigs stably expressed the transgene in a promoter-dependent manner in SB transgenic animals and only one case of variegated reporter expression was observed in the PB transgenic group [106]. This can be interpreted as transposase prefers safe harbor loci for integration.…”

“…The feasibility and efficiency of transposon-mediated transgene integration into the pig genome are supported by the high proportion of born animals carrying at least one copy of the transgene ([40 %) [65,66,106]. Reported overall efficiency was also very impressive reaching 5.7 and 6.8 % of transgenic live pigs per microinjected zygote for PB and SB transposon systems, respectively [65,106] (Table 1). Moreover, most transposon integrations corresponded to monomeric integrations and very low incidence of passive incorporation of vector backbone or SB transposase vector sequences (Table 1).…”

Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

“…The Southern blot was performed as previously described [16]. Briefly: ear genomic DNA (10 μg) from each piglet was restriction digested with Hind III, purified by ethanol precipitation, and then separated by electrophoresis in a 0.80% agarose gel.…”

“…Currently mobile genetic elements known as transposons have emerged as an alternative active transgenesis technique [13,14], which was first implemented in rodents and now applied to pigs [15,16]. The transposases currently in use for these active transgenesis techniques are piggyBac ( PB ) and Sleeping Beauty ( SB ), which are Class II cut and paste transposase enzymes [14,17,18].…”

“…In a recent report [16] we also produced EGFP-expressing founder transgenic pigs at a high success rate of 6% (TAs/micromanipulated embryos), by the simple cytoplasmic injection (CPI) technique in combination with efficient gene transfer method mediated by a proprietary self-inactivating PB vector p m GENIE-3, which has been proven efficient for mouse transgenesis [21,22]. In the present study we investigated the growth and reproduction performance, and characterized the profiles of transgene integration, expression and transmission in transgenic pigs produced in our previous experiments by PB transposition [16]. …”